Adenovirus E1a codes for two major proteins of 289 and 243 amino acids (289R and 243R, respectively) with transforming and transcriptional regulatory activities. The E1a proteins also possess activities which negatively modulate immortalization, transformation, tumorigenesis and metastasis. We propose to use an extensive panel of E1a mutants to assess its ability to suppress metastasis of human malignant melanoma cells. The invasive potential of melanoma cells expressing the E1a mutants will be determined in vitro by their ability to invade through reconstituted basement membrane and in vivo by their ability to metastasize to form lung tumors in nude mice when injected either subcutaneously or through the tail-vein. The effect of viral E1a mutants on the ability of melanoma cells to form lung metastases will also be determined by intranasal inoculation of virus particles. To understand the mechanism of suppression of metastasis, the effect of wt and mutant E1a genes on the expression of metastasis-associated genes such as nm23, collagenases and stromelysin will be determined by mutational analysis of the promoters for nm23 and collagenase. To determine whether E1a can suppress T24 ras-mediated transformation, NIH 3T3 cells will be cotransfected with the ras oncogene and various E1a mutants. Similarly, the effect of E1a on reversal of the transformed phenotype and on tumorigenicity of ras-transformed cells will also be determined. The carboxy-terminal half of E1a plays a negative role in transformation, tumorigenesis and metastasis. These activities of E1a appear to correlate with the ability of E1a to interact with a 48 kD cellular phosphoprotein. The cellular gene coding for the 48 kD protein will be cloned by probing a human cDNA library with oligonucleotide probes designed on the basis of partial amino acid sequences or by using labeled E1a C-terminal peptide to probe a cDNA expression library. In addition, other cellular genes which may interact with the C-terminal region of E1a will also be cloned using in vivo genetic systems based on protein-protein interaction in yeast or in animal cells. We have shown that conserved region (CR) 3 of 289R plays a negative role in immortalization of primary epithelial cells. We will determine whether CR3 by itself or the full- length 289R can function as dominant suppressors of immortalization by the E1a 243R protein using inducible expression systems to initiate CR3 or 289R activity. To analyze the effect of CR3 on immortalization, we will identify and clone the genes for cellular proteins which interact with this region. The proposed studies should facilitate understanding of the mechanisms of immortalization, tumorigenesis and metastasis and also aid in designing strategies to interfere with these processes.